Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
  • G01N33/528—Atypical element structures, e.g. gloves, rods, tampons, toilet paper
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
  • A61B10/0045—Devices for taking samples of body liquids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/15—Devices for taking samples of blood
  • A61B5/150007—Details
  • A61B5/150015—Source of blood
  • A61B5/150022—Source of blood for capillary blood or interstitial fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/15—Devices for taking samples of blood
  • A61B5/150007—Details
  • A61B5/150343—Collection vessels for collecting blood samples from the skin surface, e.g. test tubes, cuvettes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/505—Containers for the purpose of retaining a material to be analysed, e.g. test tubes flexible containers not provided for above
  • B01L3/5055—Hinged, e.g. opposable surfaces
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
  • A61B10/0045—Devices for taking samples of body liquids
  • A61B10/0051—Devices for taking samples of body liquids for taking saliva or sputum samples
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
  • A61B10/0045—Devices for taking samples of body liquids
  • A61B10/007—Devices for taking samples of body liquids for taking urine samples
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
  • A61B10/0045—Devices for taking samples of body liquids
  • A61B2010/0074—Vaginal or cervical secretions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/15—Devices for taking samples of blood
  • A61B5/150007—Details
  • A61B5/150358—Strips for collecting blood, e.g. absorbent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/15—Devices for taking samples of blood
  • A61B5/150969—Low-profile devices which resemble patches or plasters, e.g. also allowing collection of blood samples for testing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/12—Specific details about materials
  • B01L2300/123—Flexible; Elastomeric
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/12—Specific details about materials
  • B01L2300/126—Paper
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/10—Devices for withdrawing samples in the liquid or fluent state
  • G01N2001/1056—Disposable (single-use) samplers

Definitions

• the invention refers to the field of the analysis of cells contained in a sample of biological fluid using a medium able to maintain the membrane integrity of the cells.
• ⁇ blood cells and/or cancer cells and/or any cell which can be isolated with regard to the other samples namely a sample of urine, saliva, pus, serous fluid, vaginal fluid, tears, or feces. They are therefore eukaryotic cells.
• other cell types can be isolated from urine or saliva sample, such as epithelial cells or even parasites such as protist species.
• A“cancer cell” refers to a normal cell whose genome has accumulated several alterations.
• the cancer cell becomes minimally sensitive or insensitive to the mechanisms of tissue homeostasis (mechanisms of DNA control and repair, apoptosis, etc.) and has an indefinite capacity for proliferation (immortalization).
• red blood cells are eukaryotic cells despite the absence of a nucleus. Indeed, during the differentiation of the proerythroblast into erythrocytes (or red blood cells), hemoglobin accumulates in the cytoplasm which leads to the condensing of the nucleus, which is then expelled from the cell.
• the collection of the eukaryotic cells mentioned above also makes it possible to subsequently have access to numerous components of cells, such as organelles, proteins, nucleic acids, intracellular parasites, etc.
• the analysis of the biological fluid cells is currently carried out on samples collected by means of a standard sample.
• a standard sample As regards, for example, blood, it is a sample of several milliliters collected by means of a venipuncture or arterial puncture device (needle and sampling body or butterfly needle), the content of which is stored in collection tubes.
• venipuncture or arterial puncture device needle and sampling body or butterfly needle
• the content of which is stored in collection tubes.
• they can be stored directly in sterile boxes.
• There are other techniques for collecting a biological sample but which do not make it possible to maintain the membrane integrity of the cells and therefore do not allow cell analysis.
• DBS blood sample
• fibrous support historically cotton
• blotting paper or filter paper Conventionally used fibrous supports are cotton bases.
• the cells dry, which leads to their drying and their lysis. The cells thus lose their membrane integrity making the sample unsuitable for cell analysis.
• DNA deoxyribonucleic acid
• the analysis of dried blood drops makes it possible to carry out assays of proteins, metabolites or deoxyribonucleic acid (DNA), in particular with a view to diagnosing cancers, metabolic diseases such as phenylketonuria, or genetic diseases such as sickle cell anemia or cystic fibrosis.
• LFA Lateral Flow Assays
• the sample migrates laterally by capillary action, towards a second fiber-based medium, called a“conjugated medium.”
• a“conjugated medium” The role of this medium is to accept the conjugate, to keep it stable throughout the storage period, and to release it in an efficient and reproducible manner.
• the medium is treated with particles on which are grafted antibodies or antigens on the basis of the target sought.
• the target molecule to be analyzed binds to the conjugate and the complex thus formed, then passes over a nitrocellulose membrane, within which the presence of the target molecule is detected.
• the test is chromatographic, that is to say that the result of the analysis is visual.
• a colored band indicates that the test has worked (control band) and another band colors if it is positive.
• the conjugate will be an antibody directed specifically against this antigen, i.e. an anti ⁇ -hCG antibody.
• the anti ⁇ -hCG conjugation antibody is labeled for example with a colored particle (e.g. purple latex particle).
• the antibody captures the b-hCG contained in the urine.
• the formed antigen/antibody complex ⁇ -hCG/anti ⁇ -hCG antibody - colored latex particle), as well as the anti-hCG-colored latex antibody alone, migrate laterally by capillary action.
• the formed antigen/antibody complex encounters the anti ⁇ -hCG capture antibody at the test zone to form a complex (anti ⁇ -hCG capture antibody ⁇ -hCG/anti ⁇ -hCG conjugation antibody/colored latex particle). It is the formation of this complex which allows the coloring of the test strip.
• the conjugation anti- b-hCG antibody not coupled with b-hCG, continues its migration to the control band where it encounters the anti ⁇ -hCG antibody, which in turn attaches it on the control band.
• a colored band is formed at the window, a check making it possible to validate that the test has worked well, in particular in the case where it is negative.
• nitrocellulose induces drying of the cells present in the sample to be tested. This results in a loss of the integrity of the cell membranes (plasma and nuclear membranes), which makes any cell analysis, such as, for example, the identification of cell populations, impossible.
• One problem which the invention addresses is therefore that of developing a collection device which can be used directly by the user, along the lines of“LFA” and“DBS,” and which makes it possible to preserve the membrane integrity of cells in a biological sample for subsequent cell analysis. More specifically, one problem which the invention addresses is therefore that of developing a collection, conservation and storage medium which can maintain the membrane integrity of eukaryotic cells for a time sufficient to allow for biological analyses and tests which are otherwise impossible to carry out with available technologies, namely DBS and LFA.
• a device for collecting, preserving and storing a sample of a biological fluid comprising a porous medium for collecting and maintaining the membrane integrity of the eukaryotic cells contained in the sample, and of their constituents, the medium comprising at least 80% by weight of artificial components, advantageously at least 85%, preferably at least 90%; and having a mean flow pore size (MFP) of at least 3 pm.
• MFP mean flow pore size
• MFP designates the“ Mean Flow Pore size ,” measured according to ASTM F316-03 (2011) standard.
• “eukaryotic cells” are cells having a nucleus and internal compartments delineated by membranes. These are red blood cells (or
• erythrocytes erythrocytes
• white blood cells leukocytes
• cancer cells erythrocytes
• the eukaryotic cell has an external physical appearance compatible with cellular or subcellular analyses, without having undergone membrane lysis, whether the eukaryotic cell is alive, in quiescence, or dead after collection. It does not matter that all of the cells in the biological fluid sample have retained their membrane integrity. Indeed, it suffices that a sufficient quantity of cells has retained their membrane integrity to have a representative sample and be able to carry out the desired analysis. Many technologies allowing to study cells individually can be used on this type of biological sample.
• the device according to aspects of the invention allows the membrane integrity of eukaryotic cells and their constituents to be preserved. For example, the integrity of plasma, nuclear, mitochondrial, or even lysosomal membranes is preserved.
• “sufficient quantity of cells” is at least one eukaryotic cell which has preserved the integrity of its plasma membrane.
• the cells collected are individualized.
• the term“storage” means maintaining the integrity of the cell membranes at temperatures that are adapted on the basis of the nature of the biological sample, as per the knowledge of a person skilled in the art. For example, storage can occur at room temperature (23-25 °C) or 4 °C.
• the devices described herein allow for storage of the biological sample at a room temperature (23-25 °C) while preserving the membrane integrity of the cells.
• a room temperature 23-25 °C
• the storage of a blood sample can occur at room temperature.
• the artificial component is chosen from the group comprising artificial fibers, advantageously semi-synthetic fibers and/or synthetic fibers, and artificial foams.
• “artificial” means manufactured or produced by man, that is to say not existing in the natural state.
• “semi-synthetic fibers” are fibers for which materials of natural origin have been transformed and/or regenerated after passing through a spinneret to form fibers.
• these are rayon, lyocell ® , diacetate, triacetate, or even modal fibers.
• “synthetic fibers” are fibers for which the basic chemical units have been obtained by chemical synthesis followed by the formation of fibers.
• these are fibers based on polymers such as polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polypropylene (PP), polyethylene (PE), nylon, polyvinyl chloride (PVC), aramid, polyurethane (PU), or even elastomer fibers, glass fibers and glass micro fibers.
• polymers such as polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polypropylene (PP), polyethylene (PE), nylon, polyvinyl chloride (PVC), aramid, polyurethane (PU), or even elastomer fibers, glass fibers and glass micro fibers.
• the medium is a nonwoven material.
• the devices described herein may have the advantage of use of a minimally invasive collection which can be collected directly by the user, that is to say a patient or a healthcare staff member.
• the volume of the sample taken is small, advantageously between 10 pL and 50 pL and therefore creating less inconvenience for the individual providing the collection.
• a further advantage is the preservation of the membrane integrity of eukaryotic cells for a time sufficient to allow their analysis.
• the delay can be several hours, even several days.
• Those skilled in the art know how to verify that the membrane integrity of eukaryotic cells has been preserved by performing an analysis after elution of the cells from the medium.
• the determination of the preservation of the integrity of the plasma membrane can be verified by analysis with a phase contrast microscope, by immunological labeling, for example of membrane proteins, or even by labeling using intercalating agents of the nucleic acids making it possible to characterize a membrane permeability and therefore a loss of integrity, for example propidium iodide (PI) or 4',6-diamidino-2- phenylindole (DAPI).
• PI propidium iodide
• DAPI 4',6-diamidino-2- phenylindole
• membrane permeabilization allows the entry of all types of compounds independently of the physiological mechanisms of active or passive transport.
• PI is a cationic dye and an intercalating agent of nucleic acids without basic specificity which allows it to react with DNA or RNA. This compound is excited at 488 nm, and emits fluorescence at 617 nm when it is linked to DNA.
• Another technique more specifically specific to blood or whole blood, consists in centrifugation of the sample. If a sufficient quantity of eukaryotic cells has retained its membrane integrity, a pellet is formed at the bottom of the tube after centrifugation due to the different density gradient between plasma and light cellular debris. Thus, the plasma and light cellular debris are located in the supernatant, whereas the heavier intact cells are stored in the pellet.
• the presence of a cell pellet characterizes the preservation of the membrane integrity of the cells.
• the absence of pellet characterizes the absence of membrane integrity of the cells.
• the membrane integrity of the cells contained in a blood sample can be determined either by the method of centrifugation and identification of the presence of a cell pellet, or, by an analysis under a phase contrast microscope, or by immunological labeling, or by labeling with a DNA intercalating agent.
• the membrane integrity of the cells contained in other samples can be determined either by phase contrast microscopic analysis, by immunological labeling, or by labeling with a DNA intercalating agent.
• the medium participates in, allows and/or ensures the collection and preservation of membrane integrity.
• the physical properties of the medium according to the invention in terms of mechanical strength, weight and absorbent capacity depend on several factors, among which the quantity, type and nature of the artificial components included in the medium according to the invention, preferably semi-synthetic fibers and/or synthetic fibers.
• the preservation of the membrane integrity of the cells contained in the sample of biological fluid is obtained thanks to the nature and the quantity of the fibers of the medium, preferably a nonwoven material.
• the medium of the device contains at least 80% by weight of artificial components, advantageously at least 85%, preferably at least 90%, and even more preferably at least 95% of a total weight of the medium.
• semi-synthetic fibers are selected from the group comprising: rayon fibers, lyocell ® fibers, viscose fibers, and mixtures thereof.
• the synthetic fibers are chosen from the group comprising: polyethylene terephthalate (PET) fibers, glass fibers, glass microfibers, and their mixtures.
• PET polyethylene terephthalate
• the medium contains exclusively artificial components, preferably semi-synthetic and/or synthetic fibers, and is advantageously devoid of binding agents.
• the MFP of the medium is at least 3 pm, at least 4 pm, at least 5 pm, or even at least 10 pm, advantageously between 10 pm and 200 pm, preferably between 30 pm and 180 pm, preferentially between 45 pm and 150 pm.
• the MFP parameter is a critical point to ensure the preservation of the integrity of the cells stored by the device. Indeed, if the MFP of the medium is below 3 pm, the integrity of the membrane of the cells cannot be ensured.
• the parameter corresponding to the mean flow pore size or MFP is measured according to standard ASTM F316-03 (2011).
• the measurement can be carried out by any means known to those skilled in the art, in particular a porometer. It may, for example, be a porometer of advanced permeability PMI (or PMI Advanced Perm
• the thickness of the medium according to the invention is between 100 and 2500 pm, preferably between 200 and 1200 pm.
• the thickness of the medium is measured by the TAPPI/ANSI T411 om-15 (2015) method, identical to the ASTM D645/D645M-97 (2007) standard. The only difference with this measurement method corresponds to the application of a pressure per surface of 100 KPa, instead of 50 KPa.
• This alternative to the parameter of pressure applied is presented by the thickness measurement method as being acceptable.
• “air permeability” is the air flow through the 2 surfaces of a material under a known air pressure differential. This parameter is measured by the ASTM D737-18 (2016) standard by means of a prescribed air pressure differential of 200 Pa.
• the air permeability of the medium is between 10 and 5000 L/m 2 .s, preferably between 25 and 4500 L/m 2 .s. Applicant had found that a porosity greater than 5000 L/m 2 .s decreases the recovery level of white blood cells.
• the artificial component preferably semi-synthetic fibers and/or synthetic fibers, has a moisture regain value of less than approximately 5%.
• the“moisture regain value” is measured by the ASTM D629- 15 (2015) standard.
• the artificial component preferably the semi synthetic fibers and/or the synthetic fibers, has a contact angle with water of less than approximately 15°, advantageously less than approximately 60°.
• the“contact angle with water” is measured by the ASTM D7490-13 (2013) standard.
• the artificial component according to the invention preferably semi synthetic fibers and/or synthetic fibers, has a diameter of at least 3 pm.
• the medium consists of PET fiber for 100% of its mass, the MFP advantageously being between 50 pm and 100 pm, preferably between 65 pm and 75 pm, the thickness being advantageously included between 800 pm and 1500 pm, preferably between 1050 pm and 1150 pm.
• the cohesion of the fibers can be improved by adding a binder, which can, if necessary, itself be in the form of fibers.
• a binder which can, if necessary, itself be in the form of fibers.
• the medium, preferably the nonwoven can contain up to 15% by weight of a binder, advantageously up to 10%.
• the binder is chosen from the group comprising polyvinyl alcohol (PYOH) and the latexes, preferably the latexes of acrylic styrenes.
• a particularly advantageous binder for the medium of this invention is PVOH.
• the PVOH is in the form of fibers.
• the binder is chosen from the group comprising polyvinyl alcohol (PVOH), advantageously in the form of fibers, and the latexes, preferably the latexes of acrylic styrenes.
• the medium according to the invention consists of a mixture of glass fibers and/or PET fibers in combination with PVOH.
• the medium consists of glass fibers in combination with PVOH, the MFP advantageously being between 50 pm and 150 pm, preferably between 60 pm and 125 pm, the thickness being advantageously included between 200 pm and 400 pm, preferably between 300 pm and 350 pm.
• the medium consists of glass fibers in combination with PVOH in the form of fibers, the MFP being advantageously between 40 pm and 100 pm, preferably between 60 pm and 70 pm, the thickness advantageously being between 200 pm and 500 pm, preferably between 300 pm and 350 pm.
• the medium consists of glass fibers in combination with PVOH, the MFP being advantageously between 50 pm and 200 pm, preferably between 90 pm and 130 pm, the thickness being advantageously between 150 pm and 400 pm, preferably between 200 pm and 250 pm.
• the medium consists of glass fibers in combination with PVOH and a surface active agent (or surfactant), for example, polysorbate 20, the MFP being advantageously between 50 pm and 300 pm, preferably between 90 pm and 150 pm, the thickness being advantageously between 200 pm and 450 pm, preferably between 300 pm and 350 pm.
• a surface active agent or surfactant
• the medium consists of PET fibers in combination with an acrylic binder, for example, an acrylic styrene latex, the MFP being
• the thickness being advantageously between 200 pm and 500 pm, preferably between 300 pm and 400 pm.
• the medium consists of glass fibers in combination with PVOH, the MFP being advantageously between 50 pm and 150 pm, preferably between 75 mih and 100 mhi, the thickness being advantageously between 400 mhi and 700 mhi, preferably between 550 mhi and 600 mhi.
• the medium consists of glass fibers in combination with PVOH in the form of fibers, viscose fibers and PET fibers, the MFP being
• the thickness being advantageously between 400 pm and 700 pm, preferably between 450 pm and 550 pm.
• the medium consists of glass fibers and glass micro fibers in combination with PVOH, the MFP being advantageously between 5 pm and 50 pm, preferably between 8 pm and 15 pm, the thickness advantageously being between
• the medium consists of PET fibers in combination with a tension active agent (or surfactant), for example, polysorbate 20, the MFP advantageously being between 50 pm and 150 pm, preferably between 65 pm and 85 pm, the thickness being advantageously between 200 pm and 500 pm, preferably between 350 pm and 400 pm.
• a tension active agent or surfactant
• the medium consists of glass fibers in combination with PVOH and proteins, for example, bovine serum albumin (or BSA), the MFP being advantageously between 80 pm and 200 pm, preferably between 100 pm and 140 pm, the thickness being advantageously between 200 pm and 400 pm, preferably between 300 pm and 350 pm.
• BSA bovine serum albumin
• the medium consists of glass fibers in combination with the PVOH in the form of fibers, a tension active agent (or surfactant), for example polysorbate 20, and lyocell ® , the MFP advantageously being between 80 pm and 200 pm, preferably between 115 pm and 130 pm, the thickness being advantageously between 300 pm and 500 pm, preferably between 400 pm and 450 pm.
• a tension active agent or surfactant
• the MFP advantageously being between 80 pm and 200 pm, preferably between 115 pm and 130 pm
• the thickness being advantageously between 300 pm and 500 pm, preferably between 400 pm and 450 pm.
• the medium according to the invention has a thickness of between 100 pm and 2500 pm, advantageously between 200 pm and 1200 pm.
• the medium according to the invention is impregnated with an isotonic preservative (or isotonic preservation buffer), such as, conventionally, phosphate buffered saline (PBS), possibly combined with one or more anticoagulant agents (ethylenediaminetetraacetic acid (EDTA), heparin, citrate dextrose (ACD) etc.) and/or preservatives (such as sodium azide, paraformaldehyde etc.).
• PBS phosphate buffered saline
• anticoagulant agents ethylenediaminetetraacetic acid (EDTA), heparin, citrate dextrose (ACD) etc.
• preservatives such as sodium azide, paraformaldehyde etc.
• the isotonic preservation buffer optionally combined or supplemented with preservatives and/or anticoagulants is impregnated concomitantly with the deposit of the sample of biological fluid.
• the isotonic preservation buffer optionally combined or supplemented with preservatives and/or anticoagulants is impregnated on the medium after deposit of the sample of biological fluid.
• the proportions of isotonic preservation buffer and/or anticoagulant agents and/or preservatives used are those conventionally used in the field and known to those skilled in the art.
• the volume of isotonic preservation buffer and/or anticoagulant agents and/or preservatives isotonic preservation buffer and/or anticoagulant agents and/or preservatives
• the volume of isotonic preservation buffer and/or anticoagulant agents and/or preservatives impregnated on the medium according to the invention is between 10 and 50 pL.
• the medium according to the invention by containing an isotonic preservation buffer with or without preservation additives and/or anticoagulants, makes it possible to preserve the sample for a longer time, which prolongs the possible storage time of the biological fluid sample while maintaining the membrane integrity of a sufficient number of cells to perform the desired analysis.
• the sample of biological fluid is analyzed up to 168 hours after collection, preferably up to 72 hours after collection, for example 12 hours, 24 hours, 48 hours, or even 72 hours after collection.
• the preservation of the biological sample can be reinforced by isolating the medium from the ambient air.
• the preservation of the biological sample can be reinforced by isolating the medium from light, such as ultraviolet radiation.
• the device comprises at least one physical means and/or one chemical means capable of limiting the evaporation of the biological sample and/or its alteration by light.
• the means capable of limiting evaporation and/or its alteration by light can, for example, be a film or equivalent impermeable to gases and liquids.
• the means capable of limiting evaporation and/or its alteration by light t can be a membrane that works as a barrier to water vapor such as a microporous membrane, or even a monolithic film such as a multilayer film.
• the medium in order to identify the area of collection of the medium by the patient, the medium is sandwiched between two supports, the 2 supports being perforated opposite one another to let the medium appear.
• the presence of the two supports improves the rigidity of the medium.
• the window allows the deposit of the biological fluid sample on the medium.
• one of the supports is covered with a film impermeable to gases and liquids, at least in the area facing the medium, whereas the other support is extended laterally by a flap covering at least the area of the support facing the medium, preferably the entire surface of the support.
• Most biological fluids can be collected with the devices described herein, among which there may be blood or whole blood, urine, saliva, pus, serous fluid, vaginal fluid, tears, or even feces.
• the device is particularly suitable for blood.
• the biological fluid is blood.
• aspects of the invention also relate to a method of collecting, preserving and storing a sample of biological fluid for cell analysis, comprising a step of depositing the sample of biological fluid on the medium of the device described above.
• a sample of a biological fluid is taken which is deposited directly on the medium.
• the device is then preserved and/or stored and/or transported for cellular analysis of the fluid.
• a sufficient quantity of cells is available for analysis, possibly at least one cell, for example for a subsequent genomic analysis.
• the method comprises a subsequent step of recovering the eukaryotic cells stored on the medium.
• the recovery is accomplished by elution. It may be carried out following the collection of the sample, advantageously within 12 to 168 hours.
• the eukaryotic cells thus recovered can then be analyzed.
• the method may comprise a subsequent step of analysis of the cells and/or of the constituents of the cells contained in the sample.
• the process can be carried out with most biological fluids among which blood, urine, saliva, pus, serum, vaginal fluid, tears, or even feces.
• the method is particularly suitable for blood.
• the sample is a blood sample.
• the preservation of the membrane integrity of the eukaryotic cells allows not only the analysis of the cells, as such, but also the analysis of most of their constituents.
• the constituents are chosen from:
• mRNA messenger ribonucleic acid
• micro-RNA ii. micro-RNA (miRNA)
• HBs Hemoglobin
• Intracellular bacteria e. and all intracellular molecules or all interesting organelles, whether endogenous or exogenous.
• FIG 1 shows an exploded perspective view of a collection device according to the invention.
• FIG 2 represents the method of taking and collecting samples for cell analysis
• FIG. 3 is a photograph by phase contrast microscopy of the sample obtained by means of the device according to FIG. 1 implemented in the method of FIG. 2.
• FIG. 4 represents a two-dimensional diagram obtained after an analysis by flow cytometry, on the basis of the expression of the specific DAPI and CD45 markers, of a biological sample obtained by means of the device of the figure 1 implemented in the method of FIG. 2.
• FIG 5 represents a two-dimensional diagram obtained after an analysis by flow cytometry, on the basis of the expression of the specific markers CD3 and CD 19, of a cell population.
• FIG 6 represents a two-dimensional diagram obtained after an analysis by flow cytometry, on the basis of the expression of the specific CD4 and CD8 markers, of a cell population.
• FIG 7 represents a two-dimensional diagram obtained after an analysis by flow cytometry, on the basis of the expression of the specific EpCAM and CD45 markers, of a cell population.
• FIG 8 shows, in table form, the characteristics of examples of media according to the invention and counter-examples.
• Example 1 Example of a device according to an aspect of the invention
• Figure 1 illustrates a particular embodiment of a device according to an aspect of the invention in the form of a card (1), comprising a porous medium in the form of a nonwoven (2).
• the nonwoven medium (2) is sandwiched between two identical supports (3) and (4) made of cardboard.
• the supports (3) and (4) respectively have windows (31) and (41). These windows are placed opposite one another.
• the presence of windows on the support (3) allows access to the nonwoven medium (2) for deposit of the sample of biological fluid.
• the presence of windows makes it possible to recover the sample deposited on the nonwoven medium (2) by punching or by creep.
• An adhesive film (5) impermeable to gases and liquids is bonded to the external face of the support (4).
• a flap (6) is intended to be folded over the external face of the support (3) after depositing the biological sample.
• the flap (6) allows, after deposit of the sample, to enclose the nonwoven medium (2) between the adhesive film (5) and the flap (6).
• the nonwoven medium (2) and the biological sample are in a confined atmosphere isolated from air, light, and ambient humidity.
• Example 2 Implementation of the collection device and cell analysis a) Application to a blood sample As shown in FIG. 2, there is shown a method comprising several steps, namely:
• Step 1 Collection of the blood sample using a medium as described in Example 1 , for example.
• Step 2 Storage of the device at room temperature, that is approximately 23 °C.
• Step 3 Recovery of the sample by cutting out of a disc of the medium placed in a tube. Then, the cells are eluted, that is to say recovered and put in solution using an isotonic buffer, the PBS. This operation is carried out after 24 hours of storage.
• Step 4 Centrifugation of the eluates for 1 to 5 minutes between 300 and 600 g.
• the sample does not contain intact cells
• all the cell membranes are lysed, that is to say that the membrane integrity is not preserved. Centrifugation does not make it possible to obtain a cell pellet but only debris of cell constituents in suspension. In the case of a sample containing cells which have preserved their membrane integrity, the centrifugation will form a pellet consisting of the intact cells which can then be recovered for analysis. In other words and in this example, the preservation of the membrane integrity of the cells obtained from a blood sample is evaluated by centrifugation.
• Step 5 Cell analysis performed on the cell pellet obtained in step 4.
• Step 4 the cells recovered in Step 4 are suspended to be analyzed, for example by flow cytometry.
• Morphological parameters of size and granularity can also be measured and exploited by flow cytometry.
• the media 1 to 11 having the characteristics according to the invention, make it possible to preserve the membrane integrity of the blood cells for 24 hours after collection, on a wet (that is to say impregnated with isotonic preservation buffer) or dry medium (that is to say not impregnated with isotonic preservation buffer) and after storage at 4 °C or at room temperature (23-25 °C).
• the preservation of the membrane integrity of the cells of the sample extends up to 72 hours after their collection and storage at 4 °C or at room temperature (23-25 °C).
• the media 13 and 14 do not have the characteristics according to the invention (these media contain 100% natural fibers, by weight).
• the results show that these media when used in dry conditions, that is to say without impregnation with an isotonic preservation buffer, do not allow the membrane integrity of the cells to be preserved after 24 hours of storage at 4 °C or at room temperature (23-25 °C).
• the determination of the survival of the cells of the blood sample was evaluated after 72 hours of storage in a dry medium, at 4 °C or at room temperature.
• the cell pellet obtained at the end of step 4 mentioned above contains in particular red blood cells (erythrocytes) and white blood cells (leukocytes).
• Leukocytes have a nucleus, possibly multilobed. The presence of this nucleus then induces a change in phase of the light wave which makes it possible to identify them.
• the results are shown in FIG. 3.
• the dark cells correspond to red blood cells and the white cells to leukocytes. This photograph confirms that the cells have retained their membrane integrity, all the way to their shape, as shown by the biconcavity that is characteristic of red blood cells, thus making it possible to analyze them.
• the DAPF and CD45 + cells are living leukocytes and the DAPI + and CD45 + cells are dead leukocytes but which have kept their membrane integrity.
• CD3 and CD19 + B lymphocytes
• T lymphocytes are analyzed to identify the subpopulations (FIG. 6):
• CD4 and CD8 + T8 lymphocytes
• CD4 + and CD8 T4 lymphocytes.
• the different cell populations present in a sample obtained using the device according to the invention can be identified by flow cytometry.
• FIG. 7 represents the identification of red blood cells, leukocytes and cancer cells depending on whether the cells express the EpCAM (‘‘Epithelial cell adhesion molecule”) and CD45 biomarkers, in a blood sample obtained by means of the device according to the invention.
• EpCAM EpCAM
• CD45 biomarkers CD45 biomarkers
• EpCAM- and CD45 red blood cells
• EpCAM- and CD45 + leukocytes
• EpCAM + cancer cells.
• the device of the invention makes it possible to collect, preserve and store, thanks to the media which it contains, a sample of biological fluid under conditions such that the membrane integrity of the cells is preserved, thus making further cell analysis possible.

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